Technical development for the efficient analysis of all of the gene functions of various organisms is proceeding. Microarrays are used as analytical means this purpose. Microarrays are generally formed such that many nucleic acid fragments are aligned and immobilized on a solid support such as slide glass. Microarrays are used for methods that involve immobilizing a nucleic acid fragment sample complementary to a nucleic acid fragment (immobilized on a microarray) on a microarray via hybridization, followed by detection. As means for detecting the thus formed hybrids, a method using a fluorescent label or a radioactive label bound in advance to a nucleic acid fragment sample and a method using an intercalator that contains fluorogenic groups or conductive groups to be incorporated into hybrids are known, for example.
Examples of a method for preparing a microarray include a method (referred to as “on-chip method”) that involves directly synthesizing nucleic acids on a solid support surface and a method that involves immobilizing previously prepared nucleic acid fragments on a solid support surface. The former on-chip method involves performing a combination reaction (combinatorial synthesis) on many fine matrices on a solid support using protecting groups to be selectively removed by light irradiation and a photolithography technique and a solid phase synthesis technique to be used for semiconductor production, thereby achieving the simultaneous synthesis of many types of nucleic acid.
Meanwhile, the latter method involves spotting a previously prepared nucleic acid fragment sample or the like on a solid support surface and then binding and immobilizing the sample using covalent bonds or ionic bonds. Examples of such a method include a method that involves spotting a nucleic acid fragment sample on a solid support surface treated with a polycation (e.g., polylysine and polyethylene imine) with the use of a spotter provided with a microarray preparation apparatus, and then electrostatically binding and immobilizing the nucleic acid sample to the solid support using the electric charge of the sample; a method that involves synthesizing nucleic acids into which reaction active groups have been introduced in advance, spotting them on a surface-treated solid support surface using a spotter, and then immobilizing them via covalent bonds; and a method that involves introducing reaction active groups such as active ester groups that form covalent bonds with nucleic acids onto a solid support surface, and then immobilizing the nucleic acids via covalent bonds. Also, a method is known that involves electrostatically attracting nucleic acids to a solid su pport surface comprising a substrate, an electrostatic layer formed on the substrate for electrostatically attracting nucleic acids, and functional groups capable of covalently binding with the nucleic acids formed on the electrostatic layer, and then immobilizing the nucleic acids via covalent bonds (patent document 1) (JP Patent Publication (Kokai) No. 2008-292482 A).
Various spotters employing a pin system by which a pin tip is mechanically brought into contact with a solid support surface; an ink-jet system based on the principle of an inkjet printer; or the like are used for immobilizing nucleic acids on a solid support surface to form many spots. Most of the thus immobilized spots vary in shape. For example, spots may be falcated, doughnut-shaped, or spilled out. Varied spot shapes lead to varied signal intensities, resulting in data with low reliability. Therefore, to improve the analytical accuracy of microarrays, firm immobilization of nucleic acids in spots formed on a solid support surface, spot shapes and spot sizes that are as uniform as possible, and good reproducibility are desired when any apparatus is used.